The contractile mechanism in striated muscle cells is fairly well understood; strands of proteins (actin and myosin) are anchored by structures called z-disks that are rich in the protein .alpha.-actinin. During contraction, the protein strands slide relative to each other, exerting force against the z-disks and thereby contracting the cell.
The contractile mechanism of smooth muscle cells is not well understood. Smooth muscle cells lack the striations and z-disks of striated muscle cells, but do contain discrete bodies rich in actinin and strands of myosin and actin. These proteins are presumed to interact in smooth muscle cells as they interact in striated muscle cells, but this notion has not been verified, partly because of the difficulty of identifying long-range structural patterns in the placement of the actinin bodies, which do not appear to be ordered in any two-dimensional image.
It is known, however, that .alpha.-actinin is distributed through the cell in two types of discrete bodies of concentration: irregular plaques attached to the cell membrane, and small oblong bodies in cytoplasm just outside the nucleus of the cell. Some fluorescence images suggest that the oblong dense bodies occur in regular strands twisting through the cell in three dimensions. Since these oblong bodies apparently serve to anchor filaments of actin along the lines of force in the cell, the positions and orientations of the .alpha.-actinin bodies provide clues regarding how force is generated and transmitted through the cell.
Electron micrographs have been used to view some dense bodies, but the images do not provide enough information to discern long-range three-dimensional organization. Also, the cells must be fixed before making electron micrographs, and it is desirable to develop an imaging method that could be used on living cells.
Fluorescence digital imaging microscopy has been used to obtain an image of the .alpha.-actinin dense bodies in either living or fixed cells. In the fluorescence imaging system developed by the present applicant disclosed in "Analysis of Molecular Distribution in Singel Cells Using a Digital Imaging Microscope" by Fredric S. Fay, Kevin E. Fogarty and James M. Coggins and in U.S. patent application Ser. No. 07/034,777 filed on even date herewith (and now pending as File Wrapper Continuation Application Ser. No. 07/204,931, filed on May 31, 1988) entitled "Imaging Microspectrofluorimeter", fluorescence labelled antibodies specific to actinin are introduced into
Light of a particular frequency is then introduced to the cell and the fluorescence labelled dense bodies are illuminated. Fluorescence images of the illuminated bodies are acquired in two-dimensional planes by optical sectioning. Three-dimensional information is thereafter obtained by a grouping process of the optical sectioning. The three-dimensional image data is preprocessed to minimize image noise, nonuniformities in the optical system's gain, and distortion due to the optical system.
However, there is a problem of locating in the three-dimensional image all of the actinin dense bodies and determining the orientation of the oblong bodies. The oblong dense bodies are one voxel wide and about five voxels long (corresponding to a width of 0.25 .mu.m and a length of 1.25 .mu.m). The long axes of oblong bodies have been observed to lie within 30.degree. of the long axis of relaxed cells.
Attempts to locate and determine orientations of the bodies by visual inspection of the image planes proved impractical because of the large number of bodies and because of the difficulty of correlating traces of obliquely oriented bodies through multiple image planes in the presence of noise and distortion. A smudge in one plane could indicate a dense body, an out-of-focus structure from a nearby image plane, or a "hot spot" of fluorescence unrelated to the dense bodies being sought. Nevertheless, one cell was manually processed, giving positions and limited orientation data on the bodies. The data was used to create a rotating graphic model viewable from a fixed point in space but it proved too awkward for the level of interaction desired. Stereo images created by solid modelling of the 3-D image proved too abstract for stereopsis to be effective, and occlusion of data was a serious problem.